中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (27): 4265-4271.doi: 10.12307/2022.854

• 组织工程骨材料Tissue-engineered bone •    下一篇

改性3D打印钛支架对脂肪间充质干细胞成骨分化的影响

王  静1,杨九菊1,王宁宁1, 刘  超2   

  1. 1沧州医学高等专科学校口腔系,河北省沧州市  061001;2山东大学齐鲁医院口腔颌面外科,山东省济南市  250012
  • 收稿日期:2021-05-27 接受日期:2021-07-21 出版日期:2022-09-28 发布日期:2022-03-10
  • 通讯作者: 刘超,副主任医师,山东大学齐鲁医院口腔颌面外科,山东省济南市 250012
  • 作者简介:王静,女,1984年生,河北省霸州市人,回族,2013年石河子大学毕业,硕士,讲师,主要从事骨组织工程研究。
  • 基金资助:
    沧州医专自然科学课题(19Z015),项目负责人:王静

Effect of modified three-dimensional-printed titanium scaffold on osteogenic differentiation of adipose-derived mesenchymal stem cells

Wang Jing1, Yang Jiuju1, Wang Ningning1, Liu Chao2   

  1. 1Department of Stomatology, Cangzhou Medical College, Cangzhou 061001, Hebei Province, China; 2Department of Oral and Maxillofacial Surgery, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
  • Received:2021-05-27 Accepted:2021-07-21 Online:2022-09-28 Published:2022-03-10
  • Contact: Liu Chao, Associate chief physician, Department of Oral and Maxillofacial Surgery, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
  • About author:Wang Jing, Master, Lecturer, Department of Stomatology, Cangzhou Medical College, Cangzhou 061001, Hebei Province, China
  • Supported by:
    Natural Science Project of Cangzhou Medical College, No. 19Z015 (to WJ)

摘要:

文题释义:
联合改性的3D打印钛支架:3D打印技术制造的钛片经喷砂酸蚀和阳极氧化改性后在表面形成了微纳米多级复合结构,微米结构可使骨与植入体之间的嵌合力增加,利于细胞的黏附和增殖;纳米结构可通过调控细胞内信号分子通路,影响细胞行为。
钛表面钛阳极氧化处理:阳极氧化工艺是利用钛作为阳极、不锈钢等其他金属作为阴极,借助一定的电解液,通过电化学反应促使钛合金表面发生氧化反应而形成氧化膜。这层氧化膜对光线的反射和折射作用非常明显,且不同厚度的氧化膜会呈现出不同的颜色,同时具有保护性,因此是一种理想的装饰层与耐磨层,被广泛应用于建筑、航空、医学等领域。

背景:3D打印钛支架克服了传统钛支架内部结构可控性差及外形不匹配等缺陷,但由于钛的生物惰性较大,使其在植入体内后难以与周围组织快速稳定地结合。
目的:采用喷砂酸蚀和阳极氧化方法改性3D打印钛支架表面,观察其对脂肪间充质干细胞黏附、增殖和成骨分化能力的影响。
方法:采用喷砂酸蚀联合阳极氧化方法在3D打印钛支架表面构建微纳米多孔结构,扫描电镜观察其表面特征。将第3代大鼠脂肪间充质干细胞分别接种于3D打印钛支架组(A组)、3D打印钛支架+成骨诱导液(B组)、改性3D打印钛支架(C组)上,通过细胞骨架与CCK-8实验检测细胞的黏附和增殖能力,碱性磷酸酶与茜素红染色观察细胞的成骨分化能力,实时定量PCR分析细胞成骨相关基因的表达,免疫荧光染色观察细胞骨钙素与骨桥蛋白的表达。
结果与结论:①扫描电镜下可见,改性后的3D打印钛支架表面出现微米及亚微米级凹坑和沟槽,内部有直径为70-100 nm的纳米级孔隙,孔隙之间相互连通;②共聚焦显微镜下可见,A、B组细胞伪足及触角较少,尚未铺展;C组细胞完全铺展在材料表面,可见大量明显的细胞伪足和触角,紧密附着于材料表面;CCK-8实验显示,改性3D打印钛支架可促进脂肪间充质干细胞的增殖;③碱性磷酸酶与茜素红染色显示,B、C组碱细胞性磷酸酶活性与矿化水平高于A组(P < 0.05);④B、C组细胞骨钙素、RUNX2、碱性磷酸酶及Ⅰ型胶原mRNA表达均高于A组(P < 0.05),骨桥蛋白与骨钙素蛋白表达均高于A组(P < 0.05);⑤结果表明,喷砂酸蚀联合阳极氧化改性3D打印钛支架具有良好的生物相容性,可促进脂肪间充质干细胞的黏附、增殖和成骨分化。

https://orcid.org/0000-0002-7773-2213 (王静) 

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料口腔生物材料纳米材料缓释材料材料相容性;组织工程

关键词: 3D打印钛, 脂肪间充质干细胞, 黏附, 增殖, 生物相容性, 成骨蛋白, 成骨基因, 成骨分化

Abstract: BACKGROUND: The three-dimensional (3D)-printed titanium scaffold overcomes the disadvantages of traditional titanium scaffolds, such as poor controllability of the internal structure and mismatch in shape. However, due to the relatively large biological inertia of titanium, it is difficult to combine with surrounding tissues quickly and stably after implantation in the body. 
OBJECTIVE: To modify the surface of 3D-printed titanium by sandblasting acid etching and anodic oxidation and observe its effect on the adhesion, proliferation, and osteogenic differentiation of adipose-derived mesenchymal stem cells.
METHODS: Micro-nano porous structure was constructed on the surface of 3D-printed titanium by sandblasting acid etching and anodic oxidation. The surface was characterized by scanning electron microscopy. Adipose-derived mesenchymal stem cells at passage 3 were inoculated in the Ti group (group A), Ti+osteogenic induction group (group B), and modified Ti group (group C). Cytoskeleton and CCK-8 assay were used to detect cell adhision and proliferation. Alkaline phosphatase staining and Alizarin red staining were used to observe osteogenesis. Real-time PCR was applied to analyze the expression of osteogenic genes. Immunofluorescence staining was employed to observe the expression of osteocalcin and osteopontin in cells. 
RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that micro and submicron scale pits and grooves could be seen on the surface of the modified 3D-printed titanium scaffold. There were nanoscale pores with a diameter of 70-100 nm inside the scaffold and the pores were connected with each other. (2) Under a confocal microscope, there were few pseudopods and antennae in groups A and B, and they had not been spread; the cells in group C were completely spread on the surface of the material, and a large number of obvious pseudopods and antennae were visible, which were closely attached to the surface of the material. CCK-8 assay showed that modified 3D-printed titanium scaffolds could promote the proliferation of adipose-derived mesenchymal stem cells. (3) Alkaline phosphatase staining and Alizarin red staining exhibited that alkaline phosphatase activity and the mineralization ability were higher in the groups B and C than those in the group A (P < 0.05). (4) Osteocalcin, RUNX2, alkaline phosphatase, and type I collagen mRNA expression levels were higher in the groups B and C than those in the group A (P < 0.05). Osteopontin and osteocalcin protein expression levels were higher in the groups B and C than those in the group A (P < 0.05). (5) These results suggest that 3D-printed titanium scaffold modified by sandblasting acid etching and anodic oxidation has good biocompatibility and can promote the adhesion, proliferation and osteogenic differentiation of adipose-derived mesenchymal stem cells. 

Key words: 3D-printed titanium, adipose-derived mesenchymal stem ells, adhesion, proliferation, biocompatibility, osteogenic protein, osteogenic gene, osteogenic differentiation

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